Determination of ofloxacin in human aqueous humour by high-performance liquid chromatography with fluorescence detection

doi:10.1016/S0731-7085(96)01889-4

Journal of Pharmaceutical and Biomedical Analysis

Volume 15, Issue 5, February 1997, Pages 663-666

https://doi.org/10.1016/S0731-7085(96)01889-4 Get rights and content

Abstract

A reversed-phase high-performance liquid chromatographic method is described for the determination of ofloxacin in human aqueous humour; the method involves fluroescence detection (excitation at 290 nm; emission at 500 nm) after direct injection of samples. The method utilized a 100 mm × 8 mm i.d. cartridge column packed with 4 μm Novapak C₁₈ with a mobile phase methanol-acetonitrile-0.4 M citric acid (3:1:10, v/v/v) and a flow rate of 1 ml min⁻¹ at ambient temperature. The retention times for the internal standard pipemidic acid and for ofloxacin were 4.82 and 7.32 min respectively. The mean recovery (± ISD) from human aqueous humour was 103.24 ± 4.45% for ofloxacin at 1 μg ml⁻¹ (n = 6). The within-day and day-to-day RSDs at 0.1 μg ml⁻¹ and 1 μg ml⁻¹ were less than 6.71% (n = 6) and the lower limit of reliable determination corresponding to a signal-to-noise ratio of 2.5:1 was 20 ng ml⁻¹. The assay was shown to be suitable for measuring ofloxacin levels in human aqueous humour samples after topical, oral and intravenous administration.

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2018, Journal of Pharmaceutical and Biomedical Analysis
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Aqueous humor (AH) is a transparent fluid found in the anterior chamber of the eye. The circulating AH nourishes the cornea and lens and removes the metabolic waste moving through the ocular chambers and drains from the eye to the venous blood. Analysis of drugs in AH is necessary to evaluate their pharmacokinetics parameters, which may be crucial to avoid potential adverse effects. Analysis of endogenous components of AH may help to understand its physiology as well as changes evoked by pathological situation. This review describes analytical methods used for determination of pharmaceuticals and small endogenous molecules in AH, focusing on sample preparation procedures and analytical techniques. Studies on human and animal samples are included. After inspection and filtering of records found in PubMed about 100 research papers were selected to review. In these articles AH samples of human and rabbit origin were studied most often. Sample evaporation and reconstitution in smaller solvent volume was the most popular method for analyte pre-concentration. Acetonitrile, methanol or mixture of both solvents were used most often for protein precipitation.
Simultaneous quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in cornea and aqueous humor by LC-QTOF/MS after topical ocular dosing
2017, Journal of Pharmacological and Toxicological Methods
The fourth-generation fluoroquinolones are widely used as ophthalmic antimicrobials. This study aimed to validate a new analytical technique for simultaneous quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in the cornea and aqueous humor by liquid chromatography (LC) coupled to quadrupole time-of-flight mass spectrometry (QTOF/MS) at 10 min and 1 h after instillation of topical ophthalmic antimicrobial suspensions. It was used twenty-two male dogs without ocular lesions verified by ophthalmic and histologic examinations. Methanol:water (4:1) was used for the extraction procedure for cornea and acetonitrile:water (4:1) was used for aqueous humor. The chromatographic separations were carried out on a C18 column with a linear gradient of water and methanol, both containing 0.1% formic acid. The total chromatographic run time was 4 min. Mass spectrometry analyses were performed on a Xevo™ G2-S QTof tandem mass spectrometer, operated in a positive ion electrospray ionization (ESI +) mode. The retention times were approximately 1.42 min for gatifloxacin, 1.87 min for moxifloxacin, and 3.01 min for besifloxacin. No interference peak was detected for the three tested antimicrobials in samples obtained from both cornea and aqueous humor, ensuring that the peak response was exclusive to the analyte of interest. The limit of detection for the three antimicrobials was 0.11 μg/mL and the limit of quantification was 0.42 μg/mL for both cornea and aqueous humor samples. At both time points post instillation of the three antimicrobials, moxifloxacin had the highest corneal concentration and besifloxacin demonstrated the highest concentration in the aqueous humor.
Simultaneous determination of moxifloxacin and ofloxacin in physiological fluids using high performance liquid chromatography with ultraviolet detection
2016, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
Moxifloxacin was appropriately separated applying this method in standard mixtures, spiked plasma and aqueous humor samples as shown in Fig. 2. While ofloxacin was separated in standard mixture and spiked plasma samples while in aqueous humor ofloxacin gave interfering peaks at low concentrations, as reported earlier irrespective of its concentration [33] and at high concentrations the interfering peaks were separated to some extent from the drug. Various experimental parameters were optimized to select optimum stationary phase, mobile phase, mobile phase flow rate, internal standard, detector’s wavelength and column oven temperature.
A novel, sensitive and validated RP-HPLC–UV method was developed for simultaneous determination of moxifloxacin and ofloxacin using timolol maleate as internal standard in physiological fluids. Different experimental parameters were optimized and validated according to international guidelines. Complete separation of the analytes was achieved with Kromasil 100-5C18 analytical column (250 mm × 4.6 mm × 5 μm), methanol and 0.05% trifloroacetic acid (TFA) (38:62 v/v) were used as mobile phase, pumped at flow rate of 1.1 ml/min in isocratic phase, column oven temperature maintained at 45 °C and detection wavelength of 290 nm. Protein precipitation method was applied to extract the drugs from human plasma and bovine aqueous humor samples using methanol as precipitating solvent. This method is linear in concentration range of 0.018–100 μg/ml for moxifloxacin and 0.014–20 μg/ml for ofloxacin. The recoveries of the method were 97.52 and 97.39% in human plasma for MX and OFN respectively, while in aqueous humor 94.48% for MX. The LOD values in plasma were found to be 10.0 and 8.00 ng/ml for MX and OFN respectively, while their respective LOQ values were 18.0 and 14 ng/ml. In aqueous humor the LOD and LOQ for MX were 16.0 and 24 ng/ml respectively. In future, this method will be used to study the pharmacokinetic profile of moxifloxacin and ofloxacin in biological fluids and pharmaceutical products.
Study on a host-guest interaction of hydroxypropyl-β-cyclodextrin with ofloxacin
2015, Journal of Molecular Liquids
The host–guest interaction of hydroxypropyl-β-cyclodextrin (HP-β-CD) with ofloxacin (OFL) has been investigated by ultraviolet spectroscopy (UV), Fourier transform infrared spectroscopy (FT-IR) and molecular docking method. The influence of different factors affecting the inclusion interaction was discussed. The obtained results revealed that the inclusion complex of HP-β-CD with OFL was formed by non-bond interaction. The stoichiometry and inclusion constant (K) of OFL–HP-β-CD complex are 1:1 and 1300 M^− 1, respectively. The low aqueous solubility of ofloxacin limited its efficacy. By the inclusion process solubility of OFL was significantly enhanced. Several experimental conditions were optimized in order to obtain the maximum spectroscopy signal. Due to remarkable enhancement of absorbance in the presence of HP-β-CD, a sensitive spectrophotometric method for the OFL determination was developed. The linear range was 1.8–16 μg mL^− 1 with a correlation coefficient of 0.9996 and the detection limit 0.08 μg mL^− 1. The proposed method was applied for the determination of OFL in pharmaceutical preparations with RSD of 0.98% and the results were satisfactory in comparison to the official method.
A dual amplified electrochemical immunosensor for ofloxacin: Polypyrrole film-Au nanocluster as the matrix and multi-enzyme-antibody functionalized gold nanorod as the label
2013, Electrochimica Acta
Citation Excerpt :
However, the remain of these drugs in various foods of animal presents a potential danger such as toxic effect, resistant strain of bacteria, allergic hypersensitivity reaction, etc., which is harmful to human health especially to children or adolescents during the growth phase, and pregnancy or breast-feeding mothers [1,3]. Over years, different techniques such as spectrophotometry [3], HPLC [4], molecularly imprinted solid-phase extraction-LC [5], capillary electrophoresis [6], chemiluminescence [7], spectrofluorimetry [8] and microbiological assay [9] have been developed for the detection of OFL. These methods are well-proven and widely accepted; however, they are often viewed as laborious and time intensive in sample pretreatment.
In this work, an electrochemical immunosensor, basing on a dual signal amplified strategy by employing a biocompatible polypyrrole film-Au nanocluster matrix as a sensor platform and multi-enzyme-antibody functionalized gold nanorod as an electrochemical detection label, is established for sensitive detection of ofloxacin (OFL). Firstly, polypyrrole film and Au nanoclusters were progressively fabricated onto the surface of a glassy carbon electrode via electropolymerization and electrochemical deposition, respectively. Such PPy-Au nanocomposite modified electrode was used to immobilize OFL-OVA, blocked with the blocking reagent, and then associated with the corresponding antibody. Secondly, gold nanorod (GNR) was synthesized to load horseradish peroxidase (HRP) and horseradish peroxidase-secondary antibody (HRP-Ab₂), and the resulting nanostructure (multi-HRP-GNR-Ab₂) was applied as the detection label. The fabrication process of the ordered multilayer structure and immunosensor were characterized by scanning electron microscopy (SEM) and electrochemical measurements, respectively. Finally, based on a competitive immunoassay, i.e., the association ability with the corresponding antibody between the captured antigen and free OFL in the solution, the fabricated immunosensor exhibited a sensitive response to OFL in the range from 0.08 to 410 ng/mL with a detection limit of 0.03 ng/mL. The current immunosensor exhibited good sensitivity, selectivity and long-term stability. This amplification strategy shows excellent promise for food safety monitoring of other antibiotics and a potential application in other immunosensors.
Direct electrogenerated chemiluminescence detection in high-performance liquid chromatography for determination of ofloxacin
2008, Analytica Chimica Acta
Ofloxacin (OFLX) exhibited strong electrogenerated chemiluminescence (ECL) in NaNO₃ solution with a dual-electrode system when constant current was exerted. Based on this observation, a sensitive direct ECL method coupled with high-performance liquid chromatography (HPLC) separation was developed for determination of OFLX in human serum. Factors affected the ECL emission were investigated. Under the optimal conditions, the ECL intensity has a linear relationship with the concentration of OFLX in the range of 1.0 × 10⁻⁸ to 4.0 × 10⁻⁶ g mL⁻¹ and the detection limit was 4 × 10⁻⁹ g mL⁻¹ (S/N = 3). The proposed method was sensitive, simple and convenient to operate.

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Determination of ofloxacin in human aqueous humour by high-performance liquid chromatography with fluorescence detection

Abstract

J. Pharm. Biomed. Anal.

J. Chromatogr.

Drugs

Cornea

J. Antimicrob. Chemother.

Arch. Opthalmol.